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Genetic and Biochemical Characterization of the Proteins that Determine O-Specific Antigen Chain Length in Pseudomonas aeruginosa

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Title: Genetic and Biochemical Characterization of the Proteins that Determine O-Specific Antigen Chain Length in Pseudomonas aeruginosa
Author: Huszczynski, Steven
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Lam, Joseph
Abstract: The surfaces of bacteria are often decorated with complex polysaccharides such as capsule, teichoic acid, and lipopolysaccharide (LPS), which function as protective barriers and mediate interactions with the extracellular environment. LPS is the main constituent of the extracellular face of the prototypical Gram-negative bacterial outer membrane and is a major virulence factor. LPS is a tripartite molecule consisting of a lipid anchor, core oligosaccharide, and a long polysaccharide chain, termed the O antigen, which is highly variable in structure and length. Different O antigen chain lengths are important for conferring specific protective properties or facilitating certain interactions. This thesis examines the assembly of O antigen in the opportunistic pathogen Pseudomonas aeruginosa by two pathways commonly used by bacteria to synthesize cell surface glycoconjugates and defines the mechanisms that determine the length of the polymer chain. We identified the novel biosynthesis clusters of serotypes O15 and O17 and determine that the synthesis of these O antigens follows the ABC-transporter dependent pathway. Genetic characterization of these clusters revealed that the O15 and O17 antigens are regulated by molecular rulers with unique domain architectures. In serotype O13, we studied O antigen chain length regulation by Wzz proteins of the Wzx/Wzy-dependent pathway. Our investigation identified a new Wzz protein, Wzz3, which confers the production of novel O antigen chain lengths compared to the previously characterized Wzz1 and Wzz2. By constructing several recombinant proteins and performing extensive site-directed mutagenesis, we mapped important regions of these three chain length regulators and identified specific residues that are essential to the chain length mechanism. Overall, the results discussed here provide a comprehensive look at P. aeruginosa O antigen assembly, which will be applicable to the study of a range of important bacterial glycoconjugates.
URI: http://hdl.handle.net/10214/14714
Date: 2018-11
Rights: Attribution-NonCommercial-NoDerivatives 4.0 International
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Attribution-NonCommercial-NoDerivatives 4.0 International Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivatives 4.0 International